The Hh pathway activation requires the binding of Hh ligands (i.e. Shh, Ihh and Dhh) to the 12-pass membrane receptor Patched (PTCH), plus additional co-receptors. This interaction relieves the inhibitory activity of PTCH on the transducer SMO, a receptor with 7-pass transmembrane domains. In turn, SMO triggers downstream transcription factors belonging to the Gli family (Gli1, Gli2 and Gli3), which act on a set of target genes promoting cell proliferation and reducing cell differentiation. These target genes include Gli1 itself, thus auto-reinforcing the signaling strength and representing a sensitive read out of the pathway.
Hh signaling plays a crucial role in tissues development and proliferation (Ruiz i Altaba, et al. 2002; Ingham and Placzek 2006). A paradigmatic Hh-target organ is cerebellum, where Hh, secreted by Purkinje cells, keeps cerebellar granule cell progenitors (GCPs) proliferating, whereas its termination allows GCPs to exit the cell cycle and differentiate (Dahmane and Ruiz i Altaba 1999; Wallace 1999; Wechsler-Reya and Scott 1999).
Hh pathway is also critical for the maintenance and self-renewal of neural stem cells (NSCs) and for tumorigenesis. More specifically, Hh signaling sustains embryonic and postnatal NSCs of forebrain subventricular zone and hippocampus (Lai, et al. 2003; Machold, et al. 2003; Palma and Ruiz i Altaba 2004; Ahn and Joyner 2005; Palma, et al. 2005), as well as cerebellar NSCs and glioma stem cells (SCs) overexpressing a stemness gene signature (e.g. Nanog, Oct4, Sox2, CD133) (Clement, et al. 2007, Stecca and Ruiz i Altaba 2009).
Constitutive activation of this pathway is responsible for several malignancies, including MB, the most frequent childhood brain tumor (Ruiz i Altaba, et al. 2002). MB belongs to the group of embryonal neuroepithelial tumors and occurs in the cerebellum. This definition emphasizes the peculiar nature of this neoplasm, which is strictly related to the pediatric age; it has an exclusive origin in the only structure of the central nervous system (CNS) that continues its morphogenesis during postnatal life, and it takes origin from stem cells or cerebellar primitive precursor cells.
Aberrant Hh signaling occurs in MB as a consequence of genetic or epigenetic changes affecting several components of the pathway (Di Marcotullio, et al. 2006; Teglund and Toftgard 2010). Failure to switch off growth promoted by Hh signals during GCPs development is a primary event in MB development. Indeed, germline and somatic gain-of-function (SMO) or loss-of-function (PTCH and SUFU) mutations in components of the Hh signaling, all leading to activation of ligand-independent signals, are observed in human MB (Ellison et al., 2002; Taylor, et al. 2002). Further, mouse models in which heterozygous deletion of PTCH or activatory mutations of SMO genes result in tumor development confirm the idea that uncontrolled activation of the Hh pathway sustains the development of MB (Goodrich, et al. 1997; Hallahan, et al. 2004).
The management of this type of tumor requires aggressive treatments consisting in surgical resection followed by radiation and standard chemotherapy. Unfortunately, current therapies have serious adverse effects and patients with recurrent disease after primary therapy have a particularly poor prognosis. This is probably due to the presence in tumor mass of cancer stem cells that exhibit an increased resistance to conventional tumor treatment. In the last few years, several publications have suggested the Hh pathway as a ‘druggable’ therapeutic target in cancer, since its critical role in the maintenance of cancer stem cells in tumors. Experimental use of Hh antagonists has indicated that Hh suppression has an inhibitory effect on tumor growth in vivo and a number of Hh pathway inhibitors have been developed and patented (for a review see (Tremblay, et al. 2009). Most of these compounds act on and inhibit SMO activity. The natural teratogenic compound cyclopamine, the first identified SMO inhibitor, slows down the growth of tumors in various animal models, thus validating SMO as therapeutic target in the treatment of Hh-related diseases. Recently, several highly potent SMO antagonists have been described. Among these, vismodegib (GDC-0449) has been intensively tested and has demonstrated good inhibitory activity on the Hh pathway. This agent has been approved from FDA on January 2012 for the treatment of adults with metastatic basal cell carcinoma (BCC) or with locally advanced BCC and is currently undergoing phase II clinical trials for the treatment of ovarian, colorectal cancer and MB (for a review see De Smaele, et al. 2010). However, recent studies have described a potential mechanism of escape from vismodegib activity. In fact, it has been reported that after an initial response a patient with MB showed tumor regrowth within 3 months due to a SMO mutation (D473H) able to confer resistance to vismodegib. A mutation altering the corresponding murine residue (D477G) also arose in a vismodegib-resistant mouse model of MB (Yauch, et al. 2009; Dijkgraaf, et al. 2011). The development of resistance was also observed in mice exhibiting MB treated by SMO antagonist NVP-LDE225, which has also been progressed into clinical trials (Buonamici, et al. 2010). These findings demonstrate that acquired mutations in SMO can serve as a mechanism of drug resistance in human cancer and, in particular, underscore the need to identify new effective SMO inhibitors able to counteract tumor growth.
Further, SMO-independent Hh pathway activation, such as mutation in or loss of heterozigosity of SuFu gene (Suppressor of Fused homolog; SuFu is a physiological inhibitor of Gli1) (Taylor, et al. 2002), Gli gene amplification (Kinzler, et al. 1987) and Gli1 translocation (Dahlen, et al. 2004), has also been reported in MB and other tumor such as esophageal adenocarcinoma. This raises the need to identify novel drugs able to block Hh pathway downstream of SMO, such as targeting Gli1, the most powerful effector of the pathway.